RT Journal Article
SR Electronic
T1 Evaluation of NGS-based approaches for SARS-CoV-2 whole genome characterisation
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.07.14.201947
DO 10.1101/2020.07.14.201947
A1 Caroline Charre
A1 Christophe Ginevra
A1 Marina Sabatier
A1 Hadrien Regue
A1 Grégory Destras
A1 Solenne Brun
A1 Gwendolyne Burfin
A1 Caroline Scholtes
A1 Florence Morfin
A1 Martine Valette
A1 Bruno Lina
A1 Antonin Bal
A1 Laurence Josset
YR 2020
UL http://biorxiv.org/content/early/2020/07/15/2020.07.14.201947.abstract
AB Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialized kits including a hybridization capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP) and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples, with mNGS and RVOP producing the most complete genomes. For mid viral loads, 2/8 and 1/8 genomes were incomplete (<99%) with mNGS and both CleanPlex and RVOP, respectively. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques yielding complete genomes for 7/8 samples. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in two samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34-nt deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a default of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer annealing or probe-hybridizing regions and would require regular updates of the technique according to SARS-CoV-2 evolution.Competing Interest StatementThe authors have declared no competing interest.